Growth promoters for animals

ABSTRACT

A delivery system for providing growth promoters to food animals is provided and a method of promoting growth in food animals using such delivery systems is described.

This application is a division of application Ser. No. 07/981,082, filed Nov. 24, 1992 now U.S. Pat. No. 5,419,910.

FIELD OF THE INVENTION

This invention is directed to a delivery system which may be used to provide growth promoters for animals.

BACKGROUND OF THE INVENTION

Recent advances in pharmaceutical bioengineering have stimulated the interest of the food industry in new delivery systems for hormones and other growth promoters.

Steroids, and especially anabolic steroids, are widely used to promote the growth of cattle and other food animals. Such growth promotion is desirable among food producers because it maximizes the weight gain per animal per amount of food consumed.

Currently, steroid products are usually supplied to the animal in the form of a non-biodegradable, implantable steroid time-release pellet which is placed in the ear skin, part of the animal which is discarded upon slaughter. Such delivery systems have been recently reviewed (Sawyer, G. J., et al., Austral. Vet. J. 65:101-108 (1988). The pellet systems cannot provide a pulsed dose of steroid and are often reimplanted two or three times throughout the life of the animal with each implant being administered by a specially designed injector gun.

The use of pellet delivery systems has not met with complete success and they have many disadvantages. The main health concern is that misuse of the non-degradable pellet system may result in harmful levels of such steroids in the meat provided to consumers. Such misuse may arise if the pellet is misplaced. For example, if the pellet is misplaced in the neck or behind the ear instead of in the ear skin, the possibility is raised that the remains of the pellet could be ingested by the consumer. Also, a farmer may mistakenly inject more than one pellet at a time. Also, the pellets are often not successfully implanted and fall to the ground in the treatment pen.

Disadvantages of the currently available pellet implants further include their inability to allow for combination of different drugs with highly controlled release rates in the same unit, and the fact that only 40-70% of the active agent is released. Lastly, commercial implants do not offer a capability in adjusting the duration of action and because of the nondegradable polymeric excipient and the relatively low total surface area of the pellets, it is very difficult to adjust the dosage of the active agent.

Compudose^(R) is a polymeric controlled release implant device for use with beef cattle which is designed to provide for the continuous delivery of estradiol-17β for 200 or 400 days (Ferguson, T. H. et al., J. Cont. Rel 8:45-54 (1988)). The compudose^(R) implant is made by coating a nonmedicated silicone rubber core with a thin layer of silicone runner which contains micronized crystalline estradiol-17β. However, the device is not biodegradable.

Biodegradable particles for use in the delivery of steroid hormones have been described (U.S. Pat. No. 4,239,331, U.S. Pat. No. 4,489,055, U.S. Pat. No. 4,683,288, U.S. Pat. No. 4,677,191, U.S. Pat. No. 4,675,189, U.S. Pat. No. 4,530,840, U.S. Pat. No. 4,542,025 and U.S. Pat. No. 4,389,330.

However, none of the above references have suggested or disclosed a method of increasing animal growth by providing a biodegradable microparticle delivery of steroid growth promoters in food animals and a need exists for better, safer delivery systems for steroids in food animals.

SUMMARY OF THE INVENTION

With the above-described needs in mind, the present inventor set out to develop a composition which could be adapted to accomplish the sustained release of steroid growth promoters in food animals in a manner which would not only more efficiently promote the growth of such animals but which would also provide a safer delivery system for the growth promoting compounds.

The invention further provides a method of delivering growth promoters to animals in a multiphasic manner.

The invention further provides a method of delivering two or more active agents at predetermined release rates with the release of each drug being at a different rate and duration by design.

In detail, the invention provides a method of stimulating growth in an animal by treating such animals with injectable, biodegradable microparticles, wherein such microparticles are loaded with at least one growth-promoting steroid.

Populations of microparticles with different active steroids or other types of bioactive agents, such as antibiotics or vaccines can be blended to form a composite formulation.

In one embodiment, the growth-promoting steroid is selected from the group consisting of estradiol benzoate, 17β-estradiol, trenbolone acetate, zeranol, testosterone and testosterone derivatives or combinations thereof.

The invention further concerns the embodiment of the above-described method wherein the biodegradable microparticles comprise a homopolymer or copolymer of lactic/glycolic acid, polycaprolactone, polydioxonone, polyorthoesters, polyanhydides, and natural polymers including albumin, casein, and waxes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Estradiol Levels in Calves graphically presents the data from Table III.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In order to provide a clearer and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

By the term "administered" is intended any method of delivering the growth promoting microparticles of the invention to an animal, such as, for example, parenteral (intravenous, intramuscular, or subcutaneous) administration.

By the term "food animal" is intended any animal that is consumed as a source of protein in the diet of humans or other animals. Typical food animals include bovine animals, for example cattle; ovine animals, for example sheep; porcine, for example pigs; fowl, for example chickens and turkeys; rabbit, and the like.

By "promoting growth" or "increasing growth" is meant increasing the weight or weight gain of an animal by an amount not found in an animal whose growth was not being "promoted."

By "microparticles" is meant solid particles which contain an active agent, herein a growth promoting metabolic steroid, either in solution or in crystalline form. The active agent is dispersed or dissolved within the polymer which serves as the matrix of the particle.

The present invention concerns a method of increasing growth in food animals which comprises providing to such animals biodegradable microparticles loaded with a metabolic steroid. The method of the present invention provides advantages over methods known in the art such as, inter alia, an increased weight gain, a biodegradable system, an injectable system which prevents the loss of dose during treatment which often occurs with solid pellet implants, the ability to mix microparticles containing different drugs, the ability to program release (multiphasic release patterns) to give faster rates of drug release as the animal grows larger, and the ability to stimulate growth in poultry.

Further, the method of the invention provides the ability to design a unique blood hormone profile for the animal and a multiphasic hormone delivery system which provides pulsed doses of desired growth promoter, thus eliminating a need for consecutive implant treatments.

The product of the present invention offers the advantage of durations of action ranging from only 30 to 60 days to more than 200 days depending upon the type of microsphere selected. In the preferred embodiment the microspheres are designed to afford growth promotion in animals over a period of 150 to 250 days. The duration of action can be easily controlled by manipulation of the polymer composition, polymer:drug ratio and microsphere size.

Another important advantage of the present invention is that practically all of the active drug is delivered to the animal whereas with existing commercial implants only 40 to 75% of the active agent is released. Because the polymer used in the method of the invention is biodegradable, all of the entrapped drug can be released into the animal.

The preferred construction of microparticles of the invention are described in U.S. Pat. No. 4,389,330, and U.S. Pat. No. 4,530,840, fully incorporated herein by reference. The microparticles of the invention are composed of a polymer which is, preferably, either a homopolymer or copolymer of lactic or glycolic acids. Other bio-degradable polymers may be used, such as, for example, polycaprolactone, polydioxonene, polyorthoesters, polyanhydides, and natural polymers including albumin, casein, and waxes.

The present invention is directed to a method of stimulating growth in food animals by administering an injectable, long-acting biodegradable microparticle which contains a growth promoter for food animals. Suitable growth promoters include 17β-estradiol, estradiol benzoate, trenbolone acetate, zeranol, testosterone, testosterone derivatives (and especially testosterone propionate) and any combination thereof.

In a preferred embodiment, administration of the growth promoter to food animals by the method of the invention is achieved by a single administration of the growth promoter loaded microparticles and the microparticles release the active growth promoter in a constant or pulsed manner into the animal and eliminates the need for repetitive injections.

The formulation of the present invention contains a growth promoter dispersed in a microparticle matrix material. The amount of growth promoter agent incorporated in the microparticles usually ranges from less than 1 wt % to as high as 95 wt. %, preferably 10 to 50 wt. %. By weight % is meant parts of drug per parts of polymer by weight. For example, 10 wt. % would mean 10 parts drug per 90 parts polymer by weight.

The polymeric matrix material of the microparticles present invention must be a biocompatible and biodegradable polymeric material. The term biocompatible is defined as a polymeric material which is not toxic to an animal and not carcinogenic. The matrix material should be biodegradable in the sense that the polymeric material should degrade by bodily processes to products readily disposable by the body and should not accumulate in the body. The biodegraded products also should be biocompatible with the body in the sense that the polymeric matrix is compatible with the body. Suitable examples of polymeric matrix materials include poly (glycolic acid), poly(-d,l-lactic) acid, poly(l-lactic acid) and copolymers thereof, copolyoxalates, polycaprolactone, poly (lactic acid-caprolactone), and the like. Suitable polymeric materials also include waxes such as glycerol mono- and distearate and casein.

The molecular weight of the polymeric matrix material is of some importance. The molecular weight should be high enough so that it forms satisfactory polymer coatings, i.e., the polymer should be a good film former. Usually, a satisfactory molecular weight is greater than 5,000 daltons. However, since the properties of the film are also partially dependent on the particular polymeric material being used, it is very difficult to specify an appropriate molecular weight range for all polymers. The molecular weight of a polymer is also important from the point of view that molecular weight influences the biodegradation rate of the polymer. For a diffusional mechanism of drug release, the polymer should remain intact until all of the drug is released form the microparticles and then degrade. The drug can also be released from the microparticles as the polymeric excipient bioerodes. By an appropriate selection of polymeric materials a microparticle formulation can be made such that the resulting microparticles exhibit both diffusional release and biodegradation release properties. This is useful in affording multiphasic release patterns.

The microparticle product of the present invention can be prepared by any method which is capable of producing microparticles in a size range acceptable for use in an injectable composition. A preferred method of preparation is the method described in U.S. Pat. No. 4,389,330. In this method the desired growth promoting compound is dissolved or dispersed in an appropriate solvent. To the growth promoting agent-containing medium is added the polymeric matrix material in an amount relative to the active ingredient which gives a product of the desired loading of active agent. Optionally, all of the ingredients of the microparticle product can be blended in the solvent medium together. Suitable solvents for the growth promoter compound and the polymeric matrix material include organic solvents such as acetone, halogenated hydrocarbons such as chloroform, methylene chloride and the like, aromatic hydrocarbon compounds, halogenated aromatic hydrocarbon compounds, cyclic ethers, alcohols, ethyl acetate and the like. Preferred solvents for the growth promoter agent are methylene chloride or ethyl acetate.

The mixture of ingredients in the solvent is emulsified in a continuous-phase processing medium; the continuous-phase medium being such that a dispersion of microdroplets containing the indicated ingredients is formed in the continuous-phase medium. Naturally, the continuous-phase processing medium and the organic solvent must be immiscible, and most commonly is water although nonaqueous media such as xylene and toluene and synthetic oils and natural oils can be used. Usually, a surfactant is added to the continuous-phase processing medium to prevent the microparticles from agglomerating and to control the size of the solvent microdroplets in the emulsion. A preferred surfactant-dispersing medium combination is a 1 to 10 wt. % poly (vinyl alcohol) in water mixture. The dispersion is formed by mechanical agitation of the mixed materials. An emulsion can also be formed by adding small drops of the active agent-wall forming material solution to the continuous phase processing medium. The temperature during the formation of the emulsion is not especially critical but can influence the size and quality of the microparticles and the solubility of the drug in the continuous phase. Of course, it is desirable to have as little of the drug in the continuous phase as possible. Moreover, depending on the solvent and continuous-phase processing medium employed, the temperature must not be too low or the solvent and processing medium will solidify or the processing medium will become too viscous for practical purposes, or too high that the processing medium will evaporate, or that the liquid processing medium will not be maintained. Moreover, the temperature of the medium cannot be so high that the stability of the particular active agent being incorporated in the microparticles is adversely affected. Accordingly, the dispersion process can be conducted at any temperature which maintains stable operating conditions, which preferred temperature being about 30° C. to 60° C., depending upon the drug and excipient selected.

The dispersion which is formed is a stable emulsion and from this dispersion the organic solvent immiscible fluid can be partially removed in the first step of the solvent removal process. The solvent can easily be removed by common techniques such as heating, the application of a reduced pressure or a combination of both. The temperature employed to evaporate solvent from the microdroplets is not critical, but should not be that high that it degrades the growth promoting agent employed in the preparation of a given microparticle, nor should it be so high as to evaporate solvent at such a rapid rate to cause defects in the wall forming material. Generally, from 5 to 75%, preferably 1 to 25% of the solvent is removed in the first solvent removal step.

After the first stage, the dispersed microparticles in the solvent immiscible fluid medium are isolated from the fluid medium by any convenient means of separation. Thus, for example, the fluid can be decanted from the microparticle or the microparticle suspension can be filtered. Still other, various combinations of separation techniques can be used if desired.

Following the isolation of the microparticles from the continuous-phase processing medium, the remainder of the solvent in the microparticles is removed by extraction. In this step, the microparticles can be suspended in the same continuous-phase processing medium used in step one, with or without surfactant, or in another liquid. The extraction medium removes the solvent from the microparticles and yet does not dissolve the microparticles. During the extraction, the extraction medium with dissolved solvent must be removed and replaced with fresh extraction medium. This is best done on a continual basis, where the rate of extraction medium replenishment is critical. If the rate is too slow, agent crystals will protrude from the microparticles or grow in the extraction medium. Obviously, the rate of extraction medium replenishment or a given process is a variable which can easily be determined at the time the process is performed and, therefore, no precise limits for the rate must be predetermined. After the majority of the solvent has been removed from the microparticles, the microparticles are dried by exposure to air or by other conventional drying techniques such as vacuum drying, drying over a desiccant, or the like. This process is very efficient in encapsulating the growth promoting agent since core loadings of up to 80 wt. %, preferably up to 50 wt. % are obtained.

The microparticle product of the present invention is usually made up of particles of a spherical shape although sometimes the microparticles may be irregularly shaped. The microparticles can vary in size, ranging from submicron to millimeter diameters. Preferably, submicron to 250 μm, are desirable for growth promoter formulations allowing administration of the microparticles with a standard gauge needle.

The growth promoting agent bearing microparticles are obtained and stored as a dry material. Prior to administration to an animal or group of animals, the microparticles are suspended in an acceptable pharmaceutical liquid vehicle, and then the suspension is injected into the desired portion of the body of the animal.

The amount of growth promoting agent administered to the animal depends on the particular animal species, growth promoting agent, length of time of treatment, age of the animal and amount of growth promotion which is desired. Since the present invention is concerned with a unique method of promoting growth in food animals by administering growth promoters in a biodegradable microparticle formulation involving encapsulated growth promoting agents, one skilled in the art is well aware of the dosages required to treat a particular animal with a particular growth promoter. Commonly, growth promoters are administered in microgram to milligram amounts, depending upon the size of the animal and the length of treatment time.

The microparticles can be mixed by size or by type so as to provide for a delivery of growth promoters to animals in a multiphasic manner and/or in a manner which provides different growth promoters to the animal at different times, or a mixture of growth promoters to the animal at the same time. Other biologically active agents commonly administered to animals may be blended with the growth promoter formulation. For example, antibiotics, anthelmintics, vaccines, or any desired active agent, either in microparticle form or in conventional, unencapsulated form may be blended with the growth promoter and provided to an animal by the method of the invention.

For example, the method of the invention may also be used to deliver a vaccine containing antigenic material such as an antigen or mixture of antigens of bacterial, viral, or cellular origin. Especially, for example, the method of the invention may be used to deliver Pasteurella multocide, Pasteurella haemolytica, Haemophilus influenzae, Hemophilus ducreyi, Escherichia coli, Salmonella abortus ovis, and IBR-BVD-PI3 virus antigen(s) and vaccines.

The following examples further describe the materials and methods used in carrying out the invention. The examples are not intended to limit the invention in any manner.

EXAMPLES Example 1

Preparation of 20%-Loaded Estradiol Benzoate Microparticles

This example describes the preparation of 20% loaded estradiol benzoate microparticles with an 85:15 poly(d,l-lactide-co-glycolide) (PLGA) excipient.

To prepare a 150 g batch of 20% loaded estradiol benzoate microparticles, 30 g estradiol benzoate is dissolved in 706.06 g of 17% sterile-filtered 85:15 dl-PLGA (MW=135,964)/ethyl acetate solution. During dissolution, the contents of the Erlenmeyer flask are maintained at 60° C. Once the drug has totally dissolved, the drug/polymer solution is added to a 3000-ml jacketed reactor containing 144 g of 3% PVA having been saturated with 183 g of ethyl acetate stirring at 245 rpm at 55° C.

Once the embryonic microparticles have stabilized, the contents of the reaction flask are transferred to 110 liters of water stirring in two 60-liter stainless-steel beakers at 8° C. After having been stirred in the initial wash for 17 minutes, the hardened microparticles are wet sieved onto a 25-micron sieve. The retained product is a transferred to a second water wash of 50 liters stirring in a 60-liter stainless steel beaker at 8° C. After having been in the second wash for 15 minutes, the microparticles are again sieved onto a 25-micron sieve. The retained product is transferred to a third and final wash of 50 liters stirring at 8° C. After stirring in the third wash for about 4 hours, product is isolated by sieving through a sieve stack composed of 25, 45, 90 and 150 micron openings.

Each sieve is washed with liberal portions of fresh water and the product is allowed to air dry.

Example 2

Preparation of 15%-Loaded Estradiol Benzoate Microparticles

This example describes the preparation of 15% loaded estradiol benzoate microparticles with poly (d,l-lactide) excipient.

To prepare a 10 g batch of 15%-loaded estradiol benzoate microparticles, 1.5 g estradiol benzoate and 8.6 g dl-PLA, is dissolved in 35.7 g of ethyl acetate in an Erlenmeyer flask at 52° C. The drug/polymer solution is added to a 300-ml jacketed reaction flask containing 196 g of 3% aqueous polyvinyl alcohol having been saturated with 15.6 g of ethyl acetate stirring at 570 rpm at 52° C.

Once the embryonic microparticles have stabilized, the contents of the reaction flask are transferred to 8 liters of water stirring in 10-liter stainless steel beaker at 11° C. After stirring in the initial wash for approximately 4 minutes, the hardened microparticles are wet sieved onto a 25-micron sieve. Retained product is transferred to a second wash of 4 liters stirring at 11° C. After the product has stirred in the second wash for 6 hours, it is isolated by sieving through a sieve stack composed of 25, 45, 90, 150, and 212 micron openings. Each sieve is washed with liberal portions of fresh water to more effectively separate the different sizes of microparticles, and the product is allowed to air dry on sieves overnight.

Example 3

Preparation of 17β-Estradiol Microspheres

A 10-gram batch of 20%-loaded 17β-estradiol microspheres was prepared by weighing 8 g dl-PLA and 2 g 17β-estradiol into a 150 ml Erlenmeyer flask. Then 72 g of chloroform was added to 150 ml Erlenmeyer flask. Then 72 g of chloroform was added to the flask to dissolve the polymer. After the polymer was completely dissolved, 24 g of acetone was added to dissolve the drug. The flask was maintained at 56° C. during the drug/polymer dissolution stage. The solution was added to a 350 ml jacketed resin kettle containing 215 g of 5% polyvinyl alcohol stirring at 390 rpm and maintained at 56° C. After 4 minutes, the emulsion had stabilized and the soft microspheres were in the desired size range, the reactor contents were transferred into 8 liters of stirring water at 16° C. The microspheres were then sieved from the wash by a 25-micron sieve and the retained product was transferred into a second 8 liter stirring wash at 16° C. After stirring the product in the second wash for 3 hours, the particles were isolated by sieving through an 8 inch sieve stack composed of 25, 90, and 212 micron mesh. Each sieve was washed with liberal portions of fresh water to effectively separate the different sizes of microspheres. After air drying overnight, the product was collected, weighed and placed under ambient temperature vacuum to dry.

Example 4

Preparation of 50%-Loaded Trenbolone Acetate Microparticles

This example describes the preparation of 50%-loaded trenbolone acetate microparticles with an 85:15 poly(d,l-lactide-co-glycolide) excipient.

To prepare a 50 g batch of 50%-loaded trenbolone acetate microparticles, 25 g of trenbolone acetate and 25 g of 85:15 dl-PLGA (MW=135,964), are dissolved in 110 g of ethyl acetate in an Erlenmeyer flask at 52° C. The drug/polymer solution is added to a 1000-ml jacketed reaction flask containing about 400 g of 3% aqueous polyvinyl alcohol having been saturated with 23 g of ethyl acetate stirring at 340 rpm at 52° C.

Once the embryonic microparticles have stabilized, the contents of the reaction flask are transferred to 45 liters of water stirring in a 60-liter stainless steel beaker at 11° C. After having stirred in the initial wash for approximately 10 minutes, the hardened microparticles are wet sieved onto a 25-micron sieve. The product retained by the sieve is transferred to a second wash volume of 40 liters of water stirring at 11° C. After 5 hours stirring in second wash, the microparticles are isolated by sieving through a sieve stack composed of 25, 90, 150, 212 and 500 micron openings. Each sieve is washed with liberal portions of fresh water to more effectively separate the different sizes of microparticles and the product is allowed to air dry on sieves overnight.

Example 5

Preparation of 30%-Loaded Trenbolone Acetate Microparticles

This example describes the preparation of 30%-loaded trenbolone acetate microparticles with an 85:15 poly (d,l-lactide-co-glycolide) excipient.

To prepare a 60 g batch of 30%-loaded trenbolone acetate microparticles, 18 g of trenbolone acetate and 42 g of 85:15 dl-PLGA (MW=135,964) are dissolved in 161 g of ethyl acetate in an Erlenmeyer flask at 82° C. The drug/polymer solution is added to a 1000-ml jacketed reaction flask containing about 585 g 3% aqueous polyvinyl alcohol having been saturated with 30 g of ethyl acetate stirring at 300 rpm.

Once the embryonic microparticles have stabilized, the contents of the reaction flask are transferred to 50 liters of water stirring in a 60-liter stainless-steel beaker at 12° C. After having stirred in initial wash for approximately 15 minutes, the hardened microparticles are wet-sieved onto a 25-micron sieve. The product retained by the sieve is transferred to a second wash volume of 40 liters of water stirring at 12° C. After three hours stirring in second wash, the microparticles are isolated by sieving through a sieve stack composed of 25, 90, 150, 212 and 300 micron openings. Each sieve is washed with liberal portions of fresh water to more effectively separate the different sizes of microparticles, and the product is allowed to air-dry on sieves.

Example 6

Preparation of 25%-Loaded Zeranol Microparticles

The example describes the preparation of 25%-loaded zeranol microparticles with an 85:15 poly (d,l-lactide-co-glycolide) excipient.

To prepare a 5 g batch of 25%-loaded zeranol microparticles, 1.25 g of zeranol and 3.75 g of 85:15 dl-PLGA are dissolved in 33.0 g of ethyl acetate in an Erlenmeyer flask at 59° C. The drug/polymer solution is added to a 100-ml jacketed reaction flask containing about 93 g of 5% aqueous polyvinyl alcohol having been saturated with 5 g of ethyl acetate at 59° C and stirring at 300 rpm.

Once the embryonic microparticles had stabilized, the contents of the reaction flask are transferred to 3.5 liters of water stirring in a 4-liter beaker at 12° C. After having stirred in the initial wash for approximately 1/2 hour, the microparticles are wet sieved onto a 25-micron sieve. The retained product is transferred to a second wash of 3.5 liters of water at 12° C. After 3 hours in the second wash the microparticles are isolated by sieving through a sieve stack composed of 25, 45, 90, 150 and 212 micron pore openings. Each sieve is washed with liberal portions of water to more effectively separate the different sizes of microparticles, and the product is allowed to air dry on sieves.

Example 7

Preparation of 50%-Loaded Zeranol Microparticles

This example describes the preparation of 50%-loaded zeranol microparticles with a poly (d,l-lactide) excipient.

To prepare a 5 g batch of 50%-loaded zeranol microparticles 2.5 g of zeranol and 2.7 g of dl-PLA, dissolved in 63 g of ethyl acetate in an Erlenmeyer flask at 59° C. The drug/polymer solution is added to a 100 ml jacketed reaction flask containing about 90 g of 3% aqueous polyvinyl alcohol, having been saturated with 6 g of ethyl acetate at 58° C. and stirring at 200 rpm.

Once the embryonic microparticles have stabilized, the contents of the reaction flask are transferred to 4 liters of water stirring in a 4-liter beaker at 12° C. After having stirred in the initial wash for 23 minutes, the microparticles are wet sieved onto a 25-micron sieve. The product retained by the sieve is transferred to a second wash of 4 liters stirring at 90° C. After stirring for three hours in a second wash, the microparticles are isolated by sieving through a sieve stack composed of 25, 45, 90, 150 and 212 micron pore openings. Each sieve is washed with liberal portions of water, and the product is allowed to air dry on sieves.

Example 8

Effect of Injectable Microparticle Formulation on Promoting Weight Gain in Beef Cattle

Estradiol benzoate microparticles prepared by the methods described above were tested in beef calves to determine the effect on weight gain. Three groups of 22 calves each were used. An untreated control groups, T1, a test group T5, and a positive control group, T6. The test group (T5), received injections of 15 mg of estradiol benzoate microencapsulated in poly(lactide-co-glycolide excipient. The positive controls (T6) received the commercial implant Ralgro (zeranol active) 36 mg. The Hereford calves weighed less than 250 lbs at the time of treatment and the three groups were comprised of 10 male and 12 female animals per group. All calves remained with their dams during the 168-day study and then they were weaned. Calves were weighted, treatment site inspected, and other general health observations made at 28-day intervals. Animals were weighted on a portable Paul cattle scale equipped with a digital readout. The results of the study are shown in Tables I and II. The biodegradable microparticles were more effective than the commercial implants in increasing the weight of the animals during the treatment period.

                  TABLE 1                                                          ______________________________________                                         EFFECT OF INJECTABLE MICROPARTICLES                                            ON GROWTH OF HEREFORD BEEF CALVES                                                     Daily             Daily                                                        Gain at           Gain at                                                      Day 140                                                                               Gain       Day 168  Gain                                                (lbs)  Over Control                                                                              (lbs)    Over Control                                 ______________________________________                                         T1       1.66     --         1.62   --                                         Untreated                                                                      Control                                                                        T5       1.86     (12%)      1.82   (12.3%)                                    Estrogen                                                                       Micro-                                                                         spheres                                                                        T6       1.73     (4.2%)     1.70    (4.9%)                                    Ralgro                                                                         Estrogen          7.5% increase     7.1% increase                              Microparticles                                                                 vs.                                                                            Ralgro                                                                         ______________________________________                                    

                                      TABLE II                                     __________________________________________________________________________     Weight Gain of Hereford Calves Treated with biodegradable microspheres         Calf     Day 0 Wts.                                                                              D28                                                                               D56                                                                               D84                                                                               D112                                                                              Day 140 Wts                                                                             Day 168 Wts                                                                             Total                          Trt.                                                                              I.D.                                                                              Sex                                                                               1st                                                                               2nd                                                                               Avg.                                                                              WT WT WT WT 1st                                                                               2nd                                                                               Avg.                                                                              1st                                                                               2nd                                                                               Avg.                                                                              Gain                           __________________________________________________________________________     T5 11 H  155                                                                               149                                                                               152                                                                               196                                                                               238                                                                               312                                                                               360                                                                               417                                                                               418                                                                               418                                                                               464                                                                               468                                                                               466                                                                               314                               24 H  159                                                                               150                                                                               155                                                                               189                                                                               228                                                                               295                                                                               330                                                                               412                                                                               403                                                                               408                                                                               447                                                                               452                                                                               458                                                                               295                               25 H  131                                                                               124                                                                               128                                                                               169                                                                               216                                                                               288                                                                               312                                                                               399                                                                               404                                                                               402                                                                               459                                                                               461                                                                               460                                                                               333                               31 H  185                                                                               178                                                                               182                                                                               288                                                                               258                                                                               335                                                                               405                                                                        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337                               109                                                                               H  176                                                                               169                                                                               173                                                                               212                                                                               258                                                                               325                                                                               375                                                                               454                                                                               448                                                                               451                                                                               493                                                           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                                        316                                                                               366                                                                               372                                                                               369                                                                               257                               113                                                                               H  163                                                                               161                                                                               162                                                                               189                                                                               230                                                                               294                                                                               329                                                                               390                                                                               373                                                                               382                                                                               418                                                                               427                                                                               423                                                                               261                               127                                                                               H  146                                                                               141                                                                               144                                                                               174                                                                               204                                                                               274                                                                               317                                                                               384                                                                               375                                                                               380                                                                               428                                                                               434                                                                               431                                                                               288                               29 S  154                                                                               150                                                                               152                                                                               185                                                                               214                                                                               283                                                                               324                                                                               421                                                                               377                                                                               399                                                                               435                                                                               444                                                                               440                                                                               288                               33 S  180                                                                               173                                                                               177                                                                               205                                                                               256                                                                               343                                                                               484                                                                               491                                                                               480                                                                               486                                                                               526                                                                               532                                                                               529                                                                               353                               34 S  150                                                                               144                                                                               147                                                                               181                                                                               226                                                                               299                                                                               347                                                                               433                                                                               411                                                                               422                                                                               459                                                                               459                                                                               459                                                                               312                               57 S  186                                                                               180                                                                               183                                                                               217                                                                               255                                                                               333                                                                               375                                                                               462                                                                               443                                                                               453                                                                               504                                                                               495                                                                               500                                                                               317                               63 S  119                                                                               115                                                                               117                                                                               148                                                                               180                                                                               248                                                                               281                                                                               375                                                                               335                                                                               355                                                                               389                                                                               390                                                                               390                                                                               273                               80 S  168                                                                               165                                                                               167                                                                               197                                                                               237                                                                               314                                                                               359                                                                               434                                                                               420                                                                               427                                                                               486                                                                               498                                                                               492                                                                               326                               84 S  135                                                                               131                                                                               133                                                                               158                                                                               180                                                                               218                                                                               260                                                                               322                                                                               309                                                                               316                                                                               360                                                                               365                                                                               363                                                                               230                               85 S  192                                                                               195                                                                               194                                                                               226                                                                               268                                                                               346                                                                               387                                                                               479                                                                               432                                                                               456                                                                               584                                                                               508                                                                               586                                                                               313                               116                                                                               S  160                                                                               147                                                                               154                                                                               190                                                                               224                                                                               296                                                                               347                                                                               433                                                                               418                                                                               426                                                                               467                                                                               478                                                                               473                                                                               319                               121                                                                               S  206                                                                               196                                                                               201                                                                               241                                                                               279                                                                               348                                                                               394                                                                               462                                                                               459                                                                               461                                                                               508                                                                               496                                                                               582                                                                               301                            T6 1  H  168                                                                               149                                                                               155                                                                               182                                                                               205                                                                               273                                                                               327                                                                               401                                                                               396                                                                               399                                                                               449                                                                               442                                                                               446                                                                               291                               2  H  179                                                                               172                                                                               176                                                                               208                                                                               257                                                                               329                                                                               362                                                                               467                                                                               436                                                                               452                                                                               477                                                                               486                                                                               482                                                                               306                               46 H  174                                                                               176                                                                               175                                                                               202                                                                               234                                                                               305                                                                               355                                                                               411                                                                               414                                                                               413                                                                               452                                                                               455                                                                               454                                                                               279                               64 H  147                                                                               140                                                                               144                                                                               181                                                                               220                                                                               286                                                                               327                                                                               383                                                                               373                                                                               378                                                                               436                                                                               426                                                                               431                                                                               288                               66 H  127                                                                               122                                                                               125                                                                               164                                                                               202                                                                               274                                                                               329                                                                               413                                                                               391                                                                               402                                                                               454                                                                               460                                                                               457                                                                               333                               73 H  150                                                                               144                                                                               147                                                                               186                                                                               197                                                                               262                                                                               330                                                                               368                                                                               344                                                                               356                                                                               398                                                                               385                                                                               392                                                                               245                               92 H  193                                                                               190                                                                               192                                                                               223                                                                               260                                                                               333                                                                               347                                                                               447                                                                               433                                                                               440                                                                               498                                                                               493                                                                               496                                                                               304                               106                                                                               H  166                                                                               158                                                                               162                                                                               192                                                                               224                                                                               252                                                                               308                                                                               399                                                                               357                                                                               378                                                                               408                                                                               416                                                                               412                                                                               250                               131                                                                               H  139                                                                               134                                                                               137                                                                               169                                                                               199                                                                               264                                                                               311                                                                               396                                                                               361                                                                               379                                                                               414                                                                               396                                                                               405                                                                               269                               132                                                                               H  152                                                                               147                                                                               150                                                                               184                                                                               219                                                                               299                                                                               324                                                                               392                                                                               394                                                                               393                                                                               439                                                                               419                                                                               429                                                                               280                               139                                                                               H  103                                                                                98                                                                               101                                                                               127                                                                               155                                                                               215                                                                               258                                                                               278                                                                               276                                                                               277                                                                               336                                                                               326                                                                               331                                                                               231                               140                                                                               H  155                                                                               149                                                                               152                                                                               172                                                                               209                                                                               272                                                                               389                                                                               404                                                                               378                                                                               391                                                                               414                                                                               403                                                                               409                                                                               257                               12 S  131                                                                               131                                                                               131                                                                               160                                                                               197                                                                               257                                                                               300                                                                               367                                                                               355                                                                               361                                                                               403                                                                               415                                                                               409                                                                               278                               13 S  182                                                                               180                                                                               181                                                                               207                                                                               241                                                                               305                                                                               350                                                                               426                                                                               412                                                                               419                                                                               470                                                                               477                                                                               474                                                                               293                               16 S  159                                                                               154                                                                               157                                                                               186                                                                               233                                                                               299                                                                               344                                                                               429                                                                               422                                                                               426                                                                               475                                                                               478                                                                               477                                                                               320                               28 S  143                                                                               137                                                                               140                                                                               N.W.                                                                              197                                                                               254                                                                               306                                                                               406                                                                               364                                                                               385                                                                               429                                                                               428                                                                               429                                                                               289                               40 S  111                                                                               107                                                                               109                                                                               138                                                                               167                                                                               224                                                                               270                                                                               378                                                                               337                                                                               358                                                                               400                                                                               405                                                                               403                                                                               294                               56 S  172                                                                               163                                                                               168                                                                               194                                                                               220                                                                               272                                                                               308                                                                               382                                                                               379                                                                               381                                                                               422                                                                               413                                                                               418                                                                               250                               61 S  167                                                                               164                                                                               166                                                                               196                                                                               230                                                                               298                                                                               324                                                                               389                                                                               394                                                                               387                                                                               434                                                                               442                                                                               438                                                                               273                               70 S  188                                                                               188                                                                               188                                                                               207                                                                               247                                                                               311                                                                               374                                                                               468                                                                               449                                                                               459                                                                               516                                                                               522                                                                               519                                                                               331                               112                                                                               S  198                                                                               188                                                                               193                                                                               223                                                                               265                                                                               349                                                                               408                                                                               492                                                                               474                                                                               483                                                                               542                                                                               536                                                                               539                                                                               346                               119                                                                               S  151                                                                               144                                                                               148                                                                               178                                                                               212                                                                               279                                                                               306                                                                               418                                                                               382                                                                               400                                                                               424                                                                               424                                                                               424                                                                               277                            T1 18 H  151                                                                               149                                                                               150                                                                               180                                                                               209                                                                               270                                                                               328                                                                               422                                                                               394                                                                               408                                                                               439                                                                               443                                                                               441                                                                               291                               19 H  167                                                                               161                                                                               164                                                                               193                                                                               228                                                                               289                                                                               334                                                                               391                                                                               385                                                                               388                                                                               434                                                                               411                                                                               423                                                                               259                               37 H  158                                                                               159                                                                               159                                                                               184                                                                               219                                                                               292                                                                               330                                                                               390                                                                               388                                                                               389                                                                               426                                                                               413                                                                               420                                                                               261                               38 H  143                                                                               135                                                                               139                                                                               167                                                                               202                                                                               278                                                                               326                                                                               404                                                                               417                                                                               411                                                                               456                                                                               442                                                                               449                                                                               310                               69 H  113                                                                               106                                                                               110                                                                               143                                                                               172                                                                               232                                                                               269                                                                               335                                                                               327                                                                               331                                                                               388                                                                               388                                                                               388                                                                               279                               78 H  147                                                                               149                                                                               148                                                                               178                                                                               216                                                                               288                                                                               332                                                                               397                                                                               398                                                                               398                                                                               442                                                                               445                                                                               444                                                                               296                               98 H  163                                                                               160                                                                               162                                                                               197                                                                               235                                                                               297                                                                               324                                                                               423                                                                               391                                                                               407                                                                               431                                                                               423                                                                               427                                                                               266                               99 H  154                                                                               147                                                                               151                                                                               177                                                                               221                                                                               279                                                                               312                                                                               373                                                                               377                                                                               375                                                                               413                                                                               418                                                                               416                                                                               265                               102                                                                               H  222                                                                               215                                                                               219                                                                               250                                                                               301                                                                               388                                                                               414                                                                               503                                                                               500                                                                               502                                                                               544                                                                               544                                                                               544                                                                               326                               123                                                                               H  182                                                                               172                                                                               177                                                                               205                                                                               238                                                                               300                                                                               339                                                                               432                                                                               390                                                                               411                                                                               437                                                                               446                                                                               442                                                                               265                               126                                                                               H  175                                                                               168                                                                               172                                                                               200                                                                               232                                                                               307                                                                               357                                                                               418                                                                               413                                                                               416                                                                               454                                                                               442                                                                               448                                                                               277                               135                                                                               H  130                                                                               130                                                                               130                                                                               154                                                                               182                                                                               241                                                                               280                                                                               346                                                                               346                                                                               346                                                                               388                                                                               384                                                                               386                                                                               256                               9  S  202                                                                               201                                                                               202                                                                               230                                                                               269                                                                               335                                                                               381                                                                               477                                                                               443                                                                               460                                                                               488                                                                               508                                                                               498                                                                               297                               17 S  135                                                                               132                                                                               134                                                                               158                                                                               192                                                                               251                                                                               291                                                                               354                                                                               360                                                                               357                                                                               409                                                                               407                                                                               408                                                                               275                               21 S  168                                                                               158                                                                               163                                                                               192                                                                               223                                                                               294                                                                               335                                                                               414                                                                               388                                                                               401                                                                               426                                                                               434                                                                               430                                                                               267                               26 S  152                                                                               147                                                                               150                                                                               176                                                                               196                                                                               252                                                                               290                                                                               357                                                                               352                                                                               355                                                                               393                                                                               394                                                                               394                                                                               244                               47 S  160                                                                               158                                                                               159                                                                               184                                                                               205                                                                               277                                                                               307                                                                               359                                                                               365                                                                               362                                                                               405                                                                               408                                                                               407                                                                               248                               52 S  146                                                                               139                                                                               143                                                                               N.W.                                                                              189                                                                               244                                                                               296                                                                               350                                                                               345                                                                               348                                                                               397                                                                               401                                                                               399                                                                               257                               68 S  117                                                                               114                                                                               116                                                                               141                                                                               160                                                                               217                                                                               263                                                                               341                                                                               321                                                                               331                                                                               389                                                                               393                                                                               391                                                                               276                               101                                                                               S  183                                                                               180                                                                               182                                                                               198                                                                               235                                                                               306                                                                               359                                                                               413                                                                               405                                                                               409                                                                               455                                                                               468                                                                               458                                                                               276                               111                                                                               S  189                                                                               182                                                                               186                                                                               212                                                                               241                                                                               313                                                                               356                                                                               438                                                                               423                                                                               431                                                                               472                                                                               462                                                                               467                                                                               282                               137                                                                               S  177                                                                               169                                                                               173                                                                               198                                                                               219                                                                               286                                                                               315                                                                               412                                                                               374                                                                               393                                                                               403                                                                               390                                                                               397                                                                               224                            __________________________________________________________________________

Example 9

Duration of Delivery of the Active Agent--Use of a Mixture of 20%- and 30%-Loaded Microparticles to Deliver Estradiol Benzoate

Two batches, 20% loaded and 30% loaded, of estradiol benzoate microparticles were formulated in an 80/20 blend (by weight and tested) and tested in 6 Holstein bull calves to determine serum estradiol levels. Calves weighing between 92 and 116 pounds at the time of treatment were injected in the ear (3 calves) and in the leg muscle (3 calves) with the test material. A dose of 40 mg of estradiol benzoate (plus slight excess to offset syringe retention) microencapsulated in an 85:15 lactide:glycolide copolymer was given as a 1 cc suspension. The suspending fluid was a 2% aqueous solution of carboxymethyl cellulose with 1% tween 20 surfactant. Serum samples were collected for approximately 130 days. The results are shown in Table III, and FIG. 1. These serum estradiol levels correlated well with actual animal weight gain demonstrated when the efficacy of this product was tested in a feed lot trial. This particular combination of steroid and polymeric excipient provided efficacious levels of estradiol in the animals for 168 days.

                  TABLE III                                                        ______________________________________                                         Estradiol Levels                                                                            SR052-26B SR052-26B                                                            Muscle    Ear                                                     Days         (pg/ml)   (pg/ml)                                                 ______________________________________                                         -7           10        4                                                       -5           6         2                                                       -3           19        10                                                      0            6         4                                                       1            42        113                                                     3            19        51                                                      7            10        35                                                      10           17        178                                                     14           84        67                                                      17           56        422                                                     21           96        172                                                     24           95        326                                                     29           114       514                                                     31           105       762                                                     36           72        532                                                     38           75        667                                                     42           35        373                                                     45           22        318                                                     49           19        112                                                     52           26        81                                                      56           35        134                                                     59           44        104                                                     63           62        93                                                      66           87        335                                                     70           79        332                                                     73           56        445                                                     77           61        497                                                     80           39        225                                                     84           37        163                                                     87           52        151                                                     91           38        321                                                     94           28        244                                                     98           15        311                                                     101          16        105                                                     105          12        122                                                     108          14        114                                                     112          20        46                                                      115          13        58                                                      119          20        29                                                      122          21        25                                                      129          24        24                                                      ______________________________________                                    

Example 10

Duration of Delivery of the Active Agent--Use of a Mixture of 15%- and 30%-Loaded Microparticles to Deliver Estradiol Benzoate

Poly(d,l-lactide) was used to microencapsulate estradiol benzoate and the resulting product was administered to 6 Holstein calves to evaluate serum estradiol levels. Two batches, 15% and 30% loaded, were blended to afford the test material. The calves weighing between 101 and 145 pounds, were treated with 40 mg of microencapsulated estradiol benzoate intramuscularly at the base of the ear. The microparticles were suspended in an aqueous carboxymethyl-cellulose/tween 20 solution. Serum samples were collected at approximately 4-day intervals over a period of several months. The resulting data (Table IV) show that the biodegradable microparticles released the active agent for 200 days in the animals.

                  TABLE IV                                                         ______________________________________                                         Estradiol Levels                                                                           SR046-120                                                                      Ear                                                                       Day  (pg/ml)                                                            ______________________________________                                                -4   14.0                                                                      0    14.5                                                                      1    40.9                                                                      2    25.0                                                                      3    22.7                                                                      4    58.0                                                                      5    27.7                                                                      7    24.8                                                                      10   14.7                                                                      14   37.9                                                                      17   28.4                                                                      21   82.0                                                                      24   63.6                                                                      28   198.0                                                                     31   189.8                                                                     35   85.6                                                                      38   334.1                                                                     42   195.2                                                                     45   186.2                                                                     49   160.9                                                                     52   100.3                                                                     56   117.5                                                                     59   70.5                                                                      63   49.5                                                                      67   40.2                                                                      70   21.5                                                                      74   22.6                                                                      77   28.8                                                                      80   100.4                                                                     84   10.3                                                                      87   28.0                                                                      91   18.6                                                                      94   47.8                                                                      98   33.2                                                                      101  32.6                                                                      105  57.7                                                                      108  71.3                                                                      112  88.9                                                                      115  91.5                                                                      119  109.0                                                                     122  189.7                                                                     126  132.6                                                                     129  352.5                                                                     133  366.4                                                                     136  106.1                                                                     140  105.7                                                                     143  107.4                                                                     147  126.7                                                                     150  92.7                                                                      154  118.9                                                                     157  100.3                                                                     161  69.5                                                                      168  58.9                                                                      175  34.8                                                                      182  51.0                                                                      189  22.9                                                                      196  19.1                                                                      203  16.5                                                               ______________________________________                                    

Example 11

Duration of Delivery of the Active Agent--Use of a Mixture of 30%- and 50%-Loaded Microparticles to Deliver Trenbolone Acetate

Trenbolone acetate was microencapsulated in an 85:15 copolymer of lactic and glycolic acids, and the resulting product evaluated in 6 Holstein bull calves. The formulation tested was a 50/50 blend of a 30% loaded batch and a 50% loaded batch. Calves weighing between 91 and 117 pounds were injected either i.m. in the leg (3 calves) or at the base of the ear (3 calves) with 200 mg of encapsulate trenbolone acetate. The microparticles were suspended in an aqueous carboxymethylcellulose/tween 20 injection vehicle. Serum samples were collected and analyzed for trenbolone acetate at selected intervals. Table V shows that elevated serum drug levels were achieved over the 150-day test period. This formulation was shown to be efficacious in increasing the weight gain of beef calves.

                  TABLE V                                                          ______________________________________                                         Trenbolone acetate levels                                                                   SR046-100 SR046-100                                                            Ear       Muscle                                                  Days         (pg/ml)   (pg/ml)                                                 ______________________________________                                         -4           39        13                                                      -1           13        24                                                      0            26        47                                                      1            300       183                                                     2            202       104                                                     3            269       91                                                      4            233       135                                                     5            290       124                                                     7            295       147                                                     10           156       128                                                     14           146       92                                                      17           300       91                                                      21           272       88                                                      24           145       62                                                      28           130       30                                                      31           141       94                                                      35           318       43                                                      38           285       150                                                     42           219       222                                                     45           466       295                                                     49           517       231                                                     52           349       128                                                     56           273       129                                                     59           77        120                                                     63           83        86                                                      66           268       77                                                      70           106       81                                                      73           66        49                                                      77           206       113                                                     80           130       111                                                     84           151       125                                                     87           113       107                                                     91           105       84                                                      94           68        101                                                     98           61        65                                                      101          89        69                                                      105          108       57                                                      108          46        90                                                      112          103       79                                                      115          140       83                                                      119          115       68                                                      122          97        122                                                     129          18        122                                                     136          53        78                                                      143          35        73                                                      150          35        50                                                      ______________________________________                                    

Having now fully described the invention, it will be understood by those with skill in the art that the scope may be performed within a wide and equivalent range of conditions, parameters and the like, without affecting the spirit or scope of the invention or any embodiment thereof. 

What is claimed is:
 1. An injectable composition comprising microparticles, wherein said microparticles comprise a biodegradable polymeric matrix, wherein said composition comprises asteroid growth promoter microencapsulated within said polymeric matrix, and wherein said composition further comprises an unencapsulated anthelmintic.
 2. An injectable composition comprising microparticles, wherein said microparticles comprise a biodegradable polymeric matrix, wherein said composition comprises asteroid growth promoter microencapsulated within said polymeric matrix, and wherein said composition further comprises an anthelmintic within said polymeric matrix.
 3. An injectable composition comprising microparticles, wherein said microparticles comprise a biodegradable polymeric matrix, a steroid growth promoter microencapsulated within said polymeric matrix and an anthelmintic microencapsulated within said polymeric matrix, and wherein said steroid growth promoter and said anthelmintic are separately microencapsulated.
 4. A method of promoting growth in animals, said method comprising administration, in an amount sufficient to promote growth in said animals, of the composition of claim
 1. 5. A method of promoting growth in animals, said method comprising administration, in an amount sufficient to promote growth in said animals, of the composition of claim
 2. 6. A method of promoting growth in animals, said method comprising administration, in an amount sufficient to promote growth in said animals, of the composition of claim
 3. 